Mycobacterium tuberculosis resists elimination by human immune responses through incompletely- characterized mechanisms. To determine whether M. tuberculosis uses antigenic variation to evade elimination by T cell responses, we tested the hypothesis that M. tuberculosis generates escape mutants of the epitopes recognized by human T cells. We compared the sequences of 491 experimentally-verified human T cell epitopes in 21 strains of M. tuberculosis from the six global lineages of the M. tuberculosis complex, and made the surprising discovery that 468 (95%) of the known epitopes exhibit no sequence variation across these lineages, which represent strains of M. tuberculosis whose ancestors diverged >30,000 years ago. To determine whether the observed hyperconservation of T cell epitopes was simply the effect of low sequence diversity in the M. tuberculosis genome, we compared the ratio of the rates of nonsynonymous and synonymous single- nucleotide polymorphisms (dN/dS) in the epitopes compared with that in the experimentally-determined essential and nonessential genes, and found that the dN/dS of the known epitopes is the lowest in the M. tuberculosis genome. The observation that the known human T cell epitopes are hyperconserved suggests that the bacteria actually benefit from T cell recognition, but it is also consistent with the possibility that prior efforts at epitope discovery were skewed toward discovery of conserved epitopes. To test the hypothesis that there are variable T cell epitopes of M. tuberculosis, we will sequence 180 phylogenetically-diverse strains of M. tuberculosis, and identify the most diverse regions of the M. tuberculosis genome. We will then use extensive in silico analyses to identify those variable sequences that are predicted to encode human T cell epitopes. With that set of predicted epitopes, we will assay T cell responses to synthetic peptides having sequences matching those in the subject's infecting isolate, to identify which of the predicted epitopes are true targets of human T cell recognition. Since we anticipate that these efforts will lead to the discovery of a large set of variable epitopes, we describe further efforts to test the hypothesis that sequence variations in the newly-discovered variable epitopes are due to selection by human T cell responses. In studies beyond the scope of this application, we will use selected newly-discovered variable epitopes to test the hypothesis that human T cell recognition of variable epitopes is more closely associated with protective immunity than is recognition of the previously-discovered hyperconserved epitopes. Our studies are likely to have a large overall impact on the field of human immunity to tuberculosis, as they will markedly expand our knowledge of targets of human T cells in M. tuberculosis, they will enable novel studies and discoveries that are not currently possible, and they are likely to provide a pathway to more efficacious TB vaccines. PUBLIC HEALTH RELEVANCE: Tuberculosis afflicts nearly 1/3 of all humans, and kills nearly 2 million people every year. The work proposed in this project will reveal crucial new information on how the bacteria that cause tuberculosis interact with the human immune system. The findings will have an important impact on development of new vaccines against tuberculosis.